The present invention relates to a system for the direct sequencing of long-chain molecules such as DNA and RNA and proteins by passing the molecules through a nanoscale channel and measuring an electrical signal modulated by the molecule passing through the pore.
Genetic information may be encoded in a molecule of deoxyribonucleic acid (DNA) as a sequence of nucleotides: guanine, adenine, thymine, and cytosine. Discovering the sequence of these nucleotides in DNA and other similar molecules is a foundational technology in biological studies.
One promising method of sequencing is “nanopore sequencing” in which a single strand of DNA, forming half of the DNA helix, is passed through a nanoscale opening in a membrane between two reservoirs. This nanopore opening may, for example, be a biological pore, a solid-state pore, a semiconductor pore (nanochannel) or a DNA synthesized channel held in a lipid bilayer. By a driving force, e.g., an electrical potential applied across the reservoirs, an ion flow is produced between the reservoirs pulling the strand of DNA through the nanopore. As the strand passes through the nanopore, it modulates the ion current through the nanopore as a function of the size of the nucleotide, which partially obstructs the nanopore. This fluctuation in the ion current may then be analyzed to determine the nucleotide sequence.
The electrical signals produced by changes in ion current through a nanopore with different nucleotides are very small in amplitude and, most importantly, short in time span. For this reason, it can be hard to obtain reliable measurements having sufficient resolution to distinguish between different molecules in the sequence. Critically the size of the nanopore must be small to ensure orderly passage of the aligned molecule through the nanopore and to accentuate changes in ionic current. Such small sizes may be obtained by using biological pore molecules or retro percussive techniques developed by the present inventors.
U.S. Pat. Nos. 9,086,401 and 9,488,600 assigned to the assignee of the present application and hereby incorporated by reference, describe methods of analyzing the operation of nanopore ion channels using changes in impedance across a nanopore measured at radio frequencies. In these techniques the nanopore suspended in the membrane is flanked by conductor antennas that couple across the nanopore to measure changes in impedance (e.g., capacitance) caused by passage of the components of the molecule. This impedance measuring technique can provide gigahertz bandwidth representing a considerable advance in readout speed in comparison to techniques that measure ionic current or tunneling.
The necessarily small size of the nanopore suspended within a membrane can present significant challenges in positioning the antennas close to the nanopore in a stable fashion.